AGL proposes to develop a new, generally applicable method for the positional cloning of human growth suppressor genes. This approach merges the Selection of Hybrids by Affinity Capture technique (SHAC), developed at AGL, with the Suppression Subtractive Hybridization technique (SSH) and an in vitro functional screening assay to yield a new, potentially powerful method for the identification of growth suppressor sequences encoded by any chromosomal region of interest. The SEN6 locus on human chromosome 6q26-27 will serve as a test system for the development of this technology. Mutations at this locus are associated with immortalization of SV40-transformed human cells, and the wild-type allele appears to be responsible, at least in part, for the occurrence of cellular senescence. Furthermore, a variety of human tumors exhibit loss of heterozygosity in 6q26-27. Phase I will demonstrate the feasibility of the SHAC-SSH method through the isolation of 6q26-27-specific cDNA sequences, and sorting of the recovered cDNAs into like sets through cross-hybridization analysis. Phase II will: 1) facilitate the identification of SEN6 through DNA sequence and mRNA expression analyses, in vitro functional screening and chromosome mapping, and 2) address the general applicability of the SHAC-SSH method for the positional cloning of other growth suppressor genes. PROPOSED COMMERCIAL APPLICATION: SHAC-SSH, when fully developed in Phase II, will have general applicability to positional candidate gene isolation, from any chromosome region of interest. Reagent components for SHAC-SSH can be packaged as kits. The development of such kits should be very efficient since the most critical components; ie., chromosome and chromosome region-specific DNAs, are being produced on AGLs currently funded projects. In addition, the SHAC-SSH procedure can be performed as a service, or in strategic partnerships for specific gene discovery projects (e.g., SEN6), perhaps leading to the development of new diagnostics/therapeutics for genetically-linked diseases.